Feline Leukemia Virus
The oncomovirus feline leukemia virus (FeLV) is a member of the Retroviridae family of viruses. In cats, FeLV can cause lymphoma, myeloproliferative diseases, leukemia, immunodeficiency syndrome, aplastic anemia, and neurological disease. The virus is highly infectious for newborn or young kittens while older cats are resistant to even the most oncogenic strains of FeLV. Animals can be vaccinated against FeLV. For instance, early attempts to produce a vaccine for FeLV infection included the administration of live or inactivated FL74 feline lymphoma cells using a variety of adjuvants. However the efficacy of these vaccines has been disputed and many of these attempts were unsuccessful in protecting cats either against persistent viraemia or secondary lymphoma and sarcoma development. Soluble tumor cell antigen vaccines are presently commercially available and other experimental vaccines based on ISCOMs and subunits have also been reported.
The FeLV genome codes for three genes: a gag gene coding for the major structural components of the virus, an env gene which codes for the envelope glycoprotein, and a pol gene encoding the polymerase protein. The gag gene is expressed as a 65 kD polyprotein which is processed into four subunits: a p15 matrix protein, a p12 protein of unknown function, a p27 capsid protein, and a p10 nucleocapsid protein. The pol gene encodes three proteins: the protease, reverse transcriptase and the integrase. Autoprocessing by the protease portion of the gene gives rise to all three proteins of the pol region. Additionally, the protease is responsible for the processing of the gag precursor. The pol gene is expressed as a gag-pol fusion protein. The envelope gene is expressed as a 85 Kd glycoprotein, gp85, which is further processed to the disulfide bound, membrane associated 70 kD and 15 kD (p15E) complex found on the surface of the virion. This same gag-protease region has been inserted into the feline herpes virus (FHV) genome and shown to produce both the gag gene product and a gag-protease fusion protein in recombinant FHV infected cells. The protease was also active in processing the gag (p65) into its four polypeptide components.
Baculovirus Expression System
Baculoviruses are a large group of viruses which are highly virulent to specific insects but are not pathogenic to vertebrates or plants. With the advent of the baculovirus expression vector system (BEVS) a wide variety of genes of viral, fungal, bacterial, plant, and animal origin have been expressed in recombinant baculovirus. Briefly, BEVS uses expression vectors to insert heterologous genes into the baculovirus genome at a location such that the gene will be expressed under the control of the baculovirus regulatory elements. The recombinant baculovirus is allowed to infect a cultured insect cell line, where the heterologous protein is expressed. Several groups using this system have expressed the surface glycoprotein of two other retroviruses, human immunodeficiency virus and avian leukemia virus. Reportedly, the retroviral gag gene has also been expressed and been found to be assembled into virus-like particles in the infected insect cells.
Vaccination
Vaccination of mammals is nearly always done via the use of subcutaneous or intramuscular injection, with subcutaneous being the preferred route. In the case of small companion animals (dogs, cats, etc.) some vaccines are also administered intranasally and/or orally. Administration of vaccines to a host in these two distinct ways is disadvantageous as the immune system response is compartmentalized. This means that subcutaneous administration only really stimulates a systemic response whereas inoculation at a mucosal surface is geared to the stimulation of that distinct immunological department.
We describe here the construction of recombinant baculoviruses that not only express the gag, gp70, and gp85 genes of FeLV, but also assemble immature virus-like particles which are shed into the medium of insect cells coinfected with both the gag and gp85 viruses. In addition, the present invention provides means for making such viruses, and FeLV vaccines based on these recombinant viruses. The invention also includes methods of vaccinating a mammal and we have found, surprisingly, that by administration of a vaccine to both mucosal and systemic sites in the natural viral host, essentially full protection against persistent viraemia can be achieved.
Information Disclosure
Noteborn, M. H. M., et al, J. Gen. Virol. 71:2641-2648 (1990), report the expression of surface glycoprotein of avian leukemia virus in insect cells using BEVS. This reference does not suggest the recombinant baculovirus of the invention.
Wells, D. E., and Compans, R. W., Virology 176:575-586 (1990), Hu, S-L, et al, J. Virol. 61:3617-3620 (1987), and Rusche, J. R., et al, Proc. Natl. Acad. Sci. U.S.A. 84:6924-6928 (1987), report the expression of surface glycoprotein of human immunodeficiency virus (HIV) in insect cells using BEVS. These reference do not suggest the FeLV recombinant baculovirus of the invention.
Morikawa, S., et al, Virology 183:288-297 (1991), report the use of BEVS in the expression of the gag gene from the lentivirus feline immunodeficiency virus (FIV). The expressed protein assembles into virus like particles (VLPs) in infected insect cells when cotransfected as a gag sequence. Inclusion of the pol sequence abolished particle formation. The reference does not suggest the FeLV recombinant baculovirus of the invention.
Luo, L., et al, Virology 179:874-880 (1990), Gheysen, D., et al, Cell 59:103-112 (1989), Overton, H. A., et al, Virology 170:107-116 (1989), Hu, S-L, et al, J. Virol. 61:3617-3620 (1987), and Madisen, L., at al, Virology 158:248-250 (1987), report the expression of the lentivirus human immunodeficiency virus (HIV) gag protein assembled into virus-like particles in infected insect cells. The expressed protein assembles into virus like particles in infected insect cells when cotransfected as a gag sequence. However, again, including the pol sequence abolished the VLPs.
Delchambre, M., et al, EMBO 8:2653-2660 (1989), report the expression of the lentivirus simian immunodeficiency virus (SIV) gag protein assembled into virus-like particles in infected insect cells. The expressed protein assembles into virus like particles in infected insect cells when cotransfected with a gag sequence. However, including the pol sequence abolished the VLPs and, thus, this reference does not suggest the FeLV recombinant baculovirus of the invention.
Rasmussen, L., et al, Virology 178:435-451 (1990), report the expression of the lentivirus bovine immunodeficiency virus (BIV) gag protein assembled into virus-like particles in infected insect cells. The expressed protein assembles into virus like particles in infected insect cells when cotransfected with the gag sequence. However, including the pol sequence abolished the VLPs and, thus, this reference does not suggest the FeLV recombinant baculovirus of the invention.
Kimman, T. G., et al, Veterinary Immunology & Immunopathology 22:145-160 (1989) report on the effectiveness of a bovine respiratory syncytial virus vaccine given via a mucosal or intramuscular route. The report does not suggest administration at both sites in order to provide complete protection.